SNARE蛋白及其调节因子在S284L-Chrna4转基因鼠癫痫发作机制中的作用

It has been confirmed that exocytosis mechanism is responsible for neurotransmitter release, which is triggered by depolarization of the nerve terminal induced by an influx of Ca2+ through voltage-sensitive Ca2+ channels (VSCCs). The activation of some protien kinases by elevated Ca2+ can influence the assembly/disassembly of SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptors), which mediate the docking of vesicles and exocytosis of neurotransmitter during synaptic transmission. Recently, several studies have revealed an ipsilateral hippocampal accumulation of SNARE complexes in kindling-induced epilepsy. Interestingly, studies showed that this accumulation of SNARE complexes was correlated with enhanced glutamate release, and reversed by antiepileptic drug. Moreover, expression changes of some regulators of SNAREs in hippocampus of kindled animals were also reported. In this light, SNAREs and its regulators may be involved in the epileptic mechanisms by influencing neurotransmitter releases. Recently, we have generated transgenic rats that bear transgenes with a rat Chrna4 missense S284L mutation, corresponding to a mutation identified in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The transgenic rats, free of biological and behavioral abnormalities, showed epileptic seizure phenotypes similar to those in human ADNFLE. We have found that the dysfunction of GABA and glutamate release may play a central role in the epileptic mechanisms of ADNFLE; however, the detailed mechanisms should be clarified. Therefore, to explore the epileptic mechanisms of ADNFLE and novel targeting mechanisms for the antiepileptic actions of antiepileptic drugs, we propose to analyze the expression and function changes of SNAREs and its regulators in different brain regions of S284L-Chrna4 transgenic rats.

人类常染色体显性夜发性额叶癫痫(ADNFLE)的发病与烟碱型乙酰胆碱受体亚基的编码基因突变有关。申请者等此前已成功的开发了与ADNFLE具有同等变异基因背景的转基因动物模型(S284L-Chrna4转基因鼠)。初步研究表明S284L-Chrna4转基因鼠的癫痫发作与谷氨酸(glutamate)和GABA神经传递异常有关。为进一步阐明ADNFLE的发病机制，本课题拟利用S284L-Chrna4转基因鼠建立模拟人类癫痫疾患的动物模型平台，运用分子生物学、药理学等方法探究参与glutamate和GABA等神经递质释放机制的SNARE蛋白及其调节因子在癫痫发作机制中的作用。同时，本课题还将利用此动物模型平台探讨SNARE蛋白及其调节因子在抗癫痫药物的药理机制中的作用，为研制新型抗癫痫药物或开发新的治疗方法积累必要的基础学数据和奠定科学的理论基础。

为探讨癫痫的发病机制，本项目构建了与人类癫痫疾患具有同等遗传背景的ADNFLE转基因动物模型“S284L-Chrna4转基因鼠”，并探讨了参与谷氨酸和GABA等神经递质释放机制的SNARE蛋白及其调节因子在转基因鼠癫痫发作及在抗癫痫药药理机制中的作用。研究结果发现，转基因鼠痉挛发作期及发作间歇期脑电图呈现频发的棘锐波，发作焦点为额叶，卡马西平等抗癫痫药物能够有效地控制癫痫发作频率及抑制发作间期的异常放电。转基因鼠与正常对照之间在第4周额叶组织中与Na-K-Cl共转运体1(slc12a2, NKCC1), K-Cl共转运体1 (slc12a4, KCC1), K-Cl共转运体2 (slc12a5,KCC2),阴离子交换蛋白2(slc4a2, AE2),阴离子交换蛋白3 (slc4a3,AE3) 和阴离子交换蛋白8(slc4a8, NBCBE)mRNA表达水平无明显差异，在癫痫发作的第8周KCC1和KCC2表达减少,NKCC1表达升高，AE2, AE3, NBCBE基因表达无变化。与正照鼠比较转基因鼠神经元显示高振幅和高频率的mEPSCs，而两组间在mIPSCs的振幅和频率上无明显差异。给予4-8周转基因鼠口服呋喃苯胺酸(furosemide)，明显预防性地抑制转基因鼠在8,10,12周的癫痫发作频率及24小时脑波发作频率。电生理结果显示furosemide治疗能够明显地减少mEPSCs振幅，而不减少mEPSCs频率及mIPSCs振幅和频率。Furosemide明显改善转基因鼠额叶KCC1和KCC2基因表达的减少，而对转基因鼠NKCC1，AE2，AE3，NBCBE的mRNA表达水平无影响。说明KCC1和KCC2所介导的兴奋性-抑制性神经系统平衡紊乱在ADNFLE癫痫的发病机制及治疗中起着关键作用。本项目对理解癫痫疾患的发病机制及抗癫痫药物的痫性发作抑制机制具有重要意义。